Various publications, including patents, published applications and scholarly articles, cited throughout the present specification are incorporated by reference herein, in their entireties. Citations not fully set forth within the specification may be found at the end of the specification.
Sucrose plays an important role in the ultimate aroma and flavor that is delivered by a coffee grain or bean. Sucrose is a major contributor to the total free reducing sugars in coffee, and reducing sugars are important flavor precursors in coffee. During the roasting of coffee grain, reducing sugars will react with amino group containing molecules in a Maillard type reaction, which generates a significant number of products with caramel, sweet and roast/burnt-type aromas and dark colors that are typically associated with coffee flavor (Russwurm, 1969; Holscher and Steinhart, 1995; Badoud, 2000). The highest quality Arabica grain (Coffea Arabica) have been found to have appreciably higher levels of sucrose (between 7.3 and 11.4%) than the lowest quality Robusta grain (Coffea canephora) (between 4 and 5%) (Russwurm, 1969; Illy and Viani, 1995; Chahar et al., 2002; Badoud, 2000). Despite being significantly degraded during roasting, sucrose still remains in the roasted grain at concentrations of 0.4-2.8% dry weight (DW); thereby, contributing directly to coffee sweetness. A clear correlation exists between the level of sucrose in the grain and coffee flavor. Therefore, identifying and isolating the major enzymes responsible for sucrose metabolism and the underlying genetic basis for variations in sucrose metabolism will enable advances in the art of improving coffee quality.
Currently, there are no published reports on the genes or enzymes involved in sucrose metabolism in coffee. However, sucrose metabolism has been studied in tomato Lycopersicon esculentum (a close relative of coffee, both are members of asterid I class), especially during tomato fruit development. An overview of the enzymes directly involved in sucrose metabolism in tomato is shown in FIG. 1 (Nguyen-Quoc et al., 2001). The key reactions in this pathway are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy) and cytoplasmic invertase (I), (2) sucrose synthesis by SuSy or sucrose-phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or in the apoplast (region external to the plasma membrane, including cell walls, xylem vessels, etc) by acid invertase (vacuolar or cell wall bound) and, (4) the rapid synthesis and breakdown of starch in the amyloplast.
As in other sink organs, the pattern of sucrose unloading is not constant during tomato fruit development. At the early stages of fruit development, sucrose is unloaded intact from the phloem by the symplast pathway (direct connections between cells) and is not degraded to its composite hexoses during unloading. Both the expression and enzyme activity of SuSy are highest at this stage and are directly correlated with sucrose unloading capacity from the phloem (phenomena also called sink strength; Sun, et al., 1992; Zrenner et al., 1995). Later in fruit development, the symplastic connections are lost. Under these conditions of unloading, sucrose is rapidly hydrolyzed outside the fruit cells by the cell wall bound invertase and then the glucose and fructose products are imported into the cells by hexose transporters. Sucrose is subsequently synthesized de novo in the cytoplasm by SuSy or SPS (FIG. 1). SPS catalyses an essentially irreversible reaction in vivo due to its close association with the enzyme sucrose phosphate phosphatase (Echeverria et al., 1997). In parallel to the loss of the symplastic connections, SuSy activity decreases, and eventually becomes undetectable in fruit at the onset of ripening (Robinson et al. 1998; Wang et al. 1993). Therefore, late in the development of tomato fruit, the SPS enzyme, in association with SP, appears as the major enzymes for sucrose synthesis.
During the past decade, evidence has increasingly indicated that SuSy is responsible for the cleavage of newly imported sucrose, thereby controlling the import capacity of the fruit (N'tchobo et al., 1999) and the rate of starch synthesis. At the same time, SPS is now considered a rate limiting enzyme in the pathway providing sucrose to plant storage organs (roots, tubers and seeds) commonly referred to as sink. Together, this growing body of data strongly indicates that SuSy and SPS enzymes are important regulators of sucrose metabolism during tomato fruit development.
Alterations in carbon partitioning in plants, and most particularly improvement of sucrose levels in sink organs, have already been successfully accomplished in several plants, the most extensive and most encouraging results being obtained in tomato (Lycopersicon esculentum). Worrell and coworkers have made a set of constructions to test the effects of increasing SPS levels. For the principle experiments, they used a maize SPS cDNA under the control of the SSU promoter (Rubisco small subunit promoter) (Worrell, et al., 1991; Galtier et al. 1993; Foyer and Ferrario, 1994; Micallef, et al., 1995; Van Assche et al., 1999; Nguyen-Quoc et al., 1999). The total SPS activity in the leaves of the transformed plants was six times greater than that of the controls, while the total SPS activity in the mature fruit from the transformed plants was only twice than that of untransformed controls. This observation suggests that, even with a strong constitutive promoter, the level of recombinant SPS was altered in a tissue specific manner. Interestingly, some results have also suggested that the maize SPS activity was not under circadian control when this enzyme was expressed in tomato (Galtier et al., 1993). It should also be noted that SPS enzyme activity is negatively regulated at the post-translational level by phosphorylation and the level of phosphorylation varies according to the level of light and thus the light and dark phases of photosynthesis (Sugden et al., 1999; Jones et Ort, 1997). Therefore, the latter result suggests that the increase of SPS activity in the transgenic plants was both due to an over-expression of the protein and to the unregulated activity of the transfected maize SPS enzyme (i.e., the regulation by phosphorylation was perturbed). The increase in SPS activity was accompanied by a significant increase (25%) in total overall SuSy activity in 20 day old tomato fruit. The SuSy activity was measured with an assay in the direction of sucrose breakdown (Nguyen-Quoc et al.; 1999). Fruit from these transgenic tomato lines showed higher sugar content (36% increase) compared to untransformed plants (Van Assche et al., 1999). Biochemical studies have also shown that the high levels of the corn SPS activity in the plants caused a modification of carbohydrate portioning in the tomato leaves with an increase of sucrose/starch ratios and also a strong improvement in photosynthetic capacity. The tomato plants appeared to tolerate the elevated levels of SPS as there were no apparent detrimental growing effects. Other plants transformed with the construct 35SCaMV-SPS (35 S Cauliflower Mosaïc Virus) have three to five times more total SPS activity in leaves than in wild-type plants but surprisingly tomato fruit obtained from these particular transformants did not show any increase in SPS activity (Laporte et al. 1997; Nguyen-Quoc et al. 1999). These results indicate that the promoter selected to drive transgene expression could play an important role.
There remains a need to determine the metabolism of sucrose in coffee and the enzymes involved in the metabolism. There is also a need to identify and isolate the genes that encode these enzymes in coffee, thereby providing genetic and biochemical tools for modifying sucrose production in coffee beans to manipulate the flavor and aroma of the coffee.